LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family

Abstract

Gene licS of Bacillus subtilis encodes an excreted ␤-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two-to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in ␤-glucan utilization.Evolutionarily conserved mechanisms of gene regulation by means of transcriptional antitermination have recently been described for sucrose metabolism in Bacillus subtilis and for ␤-glucoside utilization in Escherichia coli. The ␤-glucoside (bgl) operon of E. coli consists of three genes (33) which are preceded by a catabolite gene activator protein-cyclic AMPdependent promoter (24). Interestingly, in wild-type strains, the bgl promoter is kept silent in vivo (28) and utilizes insertion sequences as transcriptional enhancers to gain full activity (24,25,31). The first gene of the operon, bglG, encodes the antiterminator protein. It is flanked by two transcriptional terminators (t1 and t2) at which the BglG protein acts to alleviate termination (20,29). Terminators t1 and t2 share a highly conserved sequence motif proximal to and extending into their stem-loop structures (33). It has been shown for terminator t1 that BglG specifically binds to this sequence motif (now termed RAT [3]) at the mRNA level (16), suggesting that binding prevents formation of the terminator structure, thereby allowing transcription to proceed.The second gene of the operon, bglF, encodes the ␤-glucoside-specific transport protein, enzyme II Bgl , which is part of the phosphoenolpyruvate sugar-phosphotransferase system (PTS) and phosphorylates its substrates concomitantly with their transport (6,32,33). Enzyme II Bgl additionally functions as negative regulator of the operon: in the absence of ␤-glucosidic substrates, it phosphorylates the BglG protein, thereby inhibiting its antiterminator activity. This phosphorylation is reversible, allowing induction of the operon upon addition of sugar substrate (1, 2, 29, 30). The third gene, bglB, codes for the hydrolyzing enzyme, a phospho-␤-glucosidase.In B. subtilis, two systems involved in sucrose metabolism are likewise controlled by transcriptional antitermi...

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT

“…The DNA sequence of a 1169 bp EcoRI fragment was determined, and this revealed the presence of an ORF encoding 281 amino acids, which is preceded by a putative ribosome-binding site (5Ј-AAAGGAGCTGA-3Ј). The deduced polypeptide encoded by this ORF has a molecular weight of 32 723, and, as observed for its C-terminal part, it is similar to transcriptional regulators of the BglG family (31% identity with BglG of E. coli, and 34%, 32% and 31% identity with the B. subtilis antiterminators SacT, SacY and LicT, respectively; Schnetz et al, 1987;Dé barbouillé et al, 1990;Zukowski et al, 1990;Schnetz et al, 1996;see Sca.GlcT, S. carnosus putative GlcT protein (Christiansen and Hengstenberg, 1996); Bsu.SacT, B. subtilis SacT (Débarbouillé et al, 1990); Bsu.SacY, B. subtilis SacY (Zukowski et al, 1990); Eco.BglG, E. coli BglG (Schnetz et al, 1987). Conserved residues are within black boxes.…”

“…In other systems regulated by transcription antitermination, the 5Ј part of the terminators overlap another inverted repeat (RAT), which is the target site of the antiterminators (Aymerich and Steinmetz, 1992;Arnaud et al, 1996). The RAT sequences are highly conserved in the E. coli bgl operon, the B. subtilis sacPA, sacXY, bglPH operons and the sacB and licS genes (Aymerich and Steinmetz, 1992; for a recent alignment of RAT sequences see Schnetz et al, 1996). The terminator of the B. subtilis pts operon overlaps an inverted repeat which is, however, dissimilar to the known RAT sequences.…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…These proteins (SacY, SacT, LicT, and GlcT) positively control the genes involved in the metabolism of carbohydrates that are taken up by the PTS; SacY and T are involved in sucrose metabolism, LicT in the metabolism of oligo--glucoside and aryl--glucoside, and GlcT in glucose assimilation. SacY and T [140][141][142] are antiterminators of the sacB (encoding levansucrase)-levB (endolevanase)-yveA, sacX (IIBC SacX )-sacY, and sacP (IIBC SacP )-sacA (sucrose-6-P hydrolase)-ywdA operons, whereas LicT 27,143) is an antiterminator of the licS gene (endo-1,3-1,4--glucanase) and the bglP (IIBCA Bgl )-bglH (6-P--glucosidase)-yxiE operon, and GlcT 144,145) is that of the ptsG (IICBA Glc )-ptsH (HPr)-ptsI (EI) operon. In the presence of the cognate PTS-sugars that activate these antiterminators, they bind to a conserved motif called the ribonucleic antiterminator (RAT), which is present in the untranslated leader-mRNAs of their target genes.…”

Systematic study of gene expression and transcription organization in the gntZ–ywaA region of the Bacillus subtilis genome

“…The red and green arrows indicate the transcripts obtained with DSM and MM, respectively. The black arrows indicate transcripts whose appearance has already been reported, these are : gntRKPZ (Fujita et al, 1986), htpG (Schulz et al, 1997), iolRS and iolABCDEFGHIJ Yoshida et al, 1997), licTS (Schnetz et al, 1996), cydABCD (Winstedt et al, 1998), licR and licBCAH (Tobisch et al, 1997) and katB (l katE) (Engelmann et al, 1995). At the top right of the figure, the graph shows the times at which the cells were harvested and the growth media for total RNA preparation.…”